N端无半胱氨酸断裂蛋白质内含子的构建  

Construction of N-terminal cysteine-free split intein

作　　者：李佳[1] 孟清[1] LI Jia;MENG Qing(Insititute of Biological Science and Biotechnology,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学生物科学与技术研究所

出　　处：《生物学杂志》2019年第4期30-34,共5页Journal of Biology

基　　金：国家自然科学基金(31570721);上海市科委基础研究重点项目(14521100700,14520720200);东华大学博士研究生创新基金(102552015094)

摘　　要：在体内或体外对目标蛋白进行标记或修饰,是研究蛋白质结构和功能的重要手段之一。目前的蛋白质修饰手段存在很多不足之处,尤其会对蛋白质的结构和功能产生影响。为了避免或降低这些影响,借助断裂蛋白质内含子反式剪接技术进行修饰是一个有效可行的方法。可是因为天然存在的断裂蛋白质非常少,使该技术的应用受到了限制。因此通过数据库筛选、定点突变、WesternBlot等技术成功获得了2个有活性的体内断裂蛋白质内含子和1个有活性的N端无半胱氨酸的断裂蛋白质内含子。为后续可控的和特异的蛋白N端标记及应用提供了可行的生物学工具。Labeling or modifying the target protein in vivo or vitro is one of the important methods to study the protein structure and function, but there are many shortcomings in current modifying methods, especially they may have the negative effect on the structure and function of the protein. To avoiding or reducing these disadvantages, modification by trans -split intein is one effective method. However, the application of the technique is limited because the nature split intein rarely exists. In this paper, two split inteins in vivo with fewer cysteines and S1-split intein in vitro with high split efficient were obtained by Inbase selection, site-directed mutations, Western Blot and other techniques. So the research supplies a reliable biology tool for controllable and specific N-terminal labeling and application of the target proteins.

关 键 词：N端无半胱氨酸断裂蛋白质内含子 蛋白质N端标记 蛋白质剪接 

分 类 号：Q816[生物学—生物工程]